Single cell full length RNA sequencing

Abstract

The invention relates to methods for processing an RNA sample and allows for single cell sequencing of full length total RNA. The method includes labeling the RNA sample with at least one of a barcode and a unique molecular identifier.

1 . A method for preparing an sequencing library, preferably a deep-sequencing library, wherein the method comprises the steps of:
 step a) providing a sample containing RNA;   step b) optionally fragmenting the RNA;   step c) polyadenylating the, optionally fragmented, RNA;   step d) hybridizing a poly-T primer to the polyadenylated RNA and performing reverse transcription of the hybridized RNA thereby obtaining cDNA; and   step e) optionally, performing a second strand synthesis,   
       wherein the poly-T primer comprises at least one of an identifier sequence (barcode) and a unique molecular identifier (UMI). 
     
     
         2 . The method according to  claim 1 , wherein the RNA sample is a cellular RNA sample, preferably wherein the RNA sample is a single cell, more preferably wherein the RNA is from a cell nucleus, most preferably from a single cell nucleus. 
     
     
         3 . The method according to  claim 1 , wherein the RNA is a small non-coding RNA, preferably selected from the group consisting of at least one of a microRNA, a snRNA and a snoRNA. 
     
     
         4 . The method according to  claim 1 , wherein the method comprises fragmentation step b), and wherein the fragmentation is step is performed by exposure to a divalent metal-cation at a temperature between about 55-100° C. 
     
     
         5 . The method according to  claim 4 , wherein the divalent metal cation is selected from the group consisting of Mg2+, Mn2+, Ca 2+ and Zn 2+, preferably the divalent metal cation is Mg2+. 
     
     
         6 . The method according to  claim 1 , wherein the method comprises fragmentation step b), and wherein the fragmentation step is followed by an end-repair step to add an OH group at the 3′end of the fragmented RNA prior to polyadenylation step c). 
     
     
         7 . The method according to  claim 1 , wherein the method further comprises at least one of the following steps:
 step f) in vitro transcription of the cDNA obtained in step d) thereby obtaining amplified RNA (aRNA); and   step g) ribosomal-RNA (rRNA) depletion.   
     
     
         8 . The method according to  claim 1 , wherein the method further comprises one or more of the following steps:
 step h) ligating an oligonucleotide adapter to the aRNA obtained in step f);   step i) performing reverse transcription of the, optionally adapter-ligated, aRNA to obtain cDNA;   step j) degrading the remaining aRNA;   step k) optionally amplifying the cDNA to generate a cDNA library comprising double-stranded cDNA with sequencing primer binding sites;   step l) selecting by size the cDNA library obtained in step k); and   step m) sequencing the, optionally size selected, cDNA library from step k).   
     
     
         9 . The method according to  claim 8 , wherein the oligonucleotide adapter in step h) comprises a barcode and optionally an UNIT 
     
     
         10 . The method according to  claim 8 , wherein the size of the size selected PCR products is between 150 bp and 1000 bp, preferably the size of the selected PCR products is between 300-450. 
     
     
         11 . The method according to  claim 7 , wherein an adapter is ligated to at least one of:
 the RNA provided in the sample of step a);   the single-stranded cDNA obtained in step d);   the double-stranded cDNA obtained in step e);   the aRNA obtained in step f); and   the cDNA obtained in step i).   
     
     
         12 . A method for obtaining RNA sequence information from a cell, preferably a single cell, comprising the steps of
 1) preparing a sequencing library as defined in  claim 1 ;   2) optionally pooling one or more sequencing libraries prepared in step 1); and   3) sequencing, preferably deep-sequencing, the sequencing library.   
     
     
         13 . A cDNA library comprising a barcode and a UMI obtainable by the methods of  claim 1 . 
     
     
         14 . The cDNA library according to  claim 13 , wherein the cDNA library is further processed to be sequenced.